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Abstract
We find that several key endogenous protein structures give rise to intense second-harmonic
generation (SHG)-nonabsorptive frequency doubling of an excitation laser line. Second-harmonic
imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful
and unique tool for high-resolution, high-contrast, three-dimensional studies of live
cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching
or toxicity and does not require exogenous labels. Unlike polarization microscopy,
SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this
study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained
thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in
a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live
cells and tissues. SHG arose not only from coiled-coil complexes within connective
tissues and muscle thick filaments, but also from microtubule arrays within interphase
and mitotic cells. Both polarization dependence and a local symmetry cancellation
effect of SHG allowed the signal from species generating the second harmonic to be
decoded, by ratiometric correlation with TPEF, to yield information on local structure
below optical resolution. The physical origin of SHG within these tissues is addressed
and is attributed to the laser interaction with dipolar protein structures that is
enhanced by the intrinsic chirality of the protein helices.